The present invention relates to the area of cancer diagnostics and therapeutics. More specifically, the invention relates to the Int6 gene and to the use of reagents derived from the nucleic acid and deduced amino acid sequences of the Int6 gene in gene therapy, vaccines, diagnostic methods and immunotherapy.
The mouse mammary tumor virus (MMTV) is a retrovirus which has been shown to act as an insertional mutagen that causes the deregulation of expression of cellular genes adjacent to the site of MMTV integration in mammary tumors (Varmus, H. E. (1982). Cancer Surv., 1:309-320). Mice infected with MMTV frequently develop preneoplastic hyperplastic alveolar nodules (HAN) (Daniel, C., et al. Proc. Natl. Acad. Sci. USA., 61:53-60; DeOme, K. B., (1959) J. Natl. Cancer Inst., 78:751-757; Medina, D., (1973) Methods Cancer Res., 7:3-53; Smith, G., et al. (1984) Cancer Res., 44:3426-3437) and the passage of these nodules in cleared mammary fat pads of syngeneic mice by serial outgrowth often results in the development of mammary tumors within these mice in a stochastic manner. In addition, it is not uncommon to find metastatic lesions in the lungs of mice bearing outgrowths with mammary tumors. For these reasons, there is considerable interest in identifying MMTV-induced mutational events that may contribute to different stages of tumor development.
Using the MMTV genome as a molecular tag, five loci (Wnt-1/Int-1, Fgf-3/Int-2, Int-3, Wnt-3, and Fgf-4/Hst/k-FGF) have been identified which represent common integration sites (designated Int loci) for MMTV in mouse mammary tumors (Dickson, C., et al. (1984) Cell., 37:529-536; Gallahan, D., et al. (1987) J. Virol., 61:218-220; Nusse, R., et al. (1982) Cell., 31:99-109; Peters, G., et al. (1989) Proc. Natl. Acad. Sci. USA., 86:5678-5682). Transgenic mouse studies utilizing transgenes in which the MMTV LTR was linked to either the WNT-1, Fgf-3, or Int-3 genes have demonstrated that activation of expression of these genes contributes to mammary tumorigenesis (Jhappan, C., et al. (1992) Genes and Develop., 6:345-355; Muller, W. J., et al. (1990) Embo J., 9:907-913; Tsukamoto, A. S., et al. (1988) Cell., 55:619-625).
The present invention describes the isolation of a new Int gene designated Int6 and the use of the gene, its gene product, and reagents derived from the gene and its gene product, in diagnostic methods, vaccines, immunotherapy and gene therapy.
The present invention relates to the isolation of the Int6 gene. The invention also relates to the murine and human cDNAs which comprise the coding sequence of the Int6 gene. The invention further relates to nucleic acid sequences derived from the Int6 gene and the Int6 cDNAs and the use of these nucleic acid sequences as probes to isolate homologues of the Int6 gene in other mammals or as probes to detect mutations of the Int6 gene.
It is also an object of the present invention to provide synthetic nucleic acid sequences capable of directing production of recombinant Int6 protein and peptide fragments derived therefrom, as well as equivalent natural nucleic acid sequences. Such natural nucleic acid sequences may be isolated from a cDNA or genomic library from which the gene capable of directing the synthesis of the Int6 protein may be identified and isolated. For the purposes of this application, nucleic acid sequence refer to RNA, DNA, cDNA or any synthetic variant thereof.
The present invention further relates to Int6 protein and peptides derived therefrom.
The invention also relates to antibodies directed against Int6 protein or peptides derived therefrom.
The invention also provides methods for detecting mutations of Int6 gene where detection of such mutations is useful in determining the presence of a neoplastic tissue in a subject or a genetic predisposition to cancer in a subject.
A first method for detecting mutations of the Int6 gene comprises analyzing DNA of a subject for mutations of the Int6 gene.
A second method for detecting mutations of the Int6 gene comprises analyzing RNA of a subject for alterations in the Int6 mRNA expression.
Yet another method for detecting mutations of the Int6 gene comprises analyzing protein of a subject for alterations in Int6 protein expression.
The present invention also provides pharmaceutical compositions for use as vaccines for immunizing a subject against cancer and for use in immunotherapy methods. One such composition comprises nucleic acid sequence capable of directing host organism synthesis of Int6 protein or peptide fragments thereof while a second pharmaceutical composition comprises Int6 protein or peptides derived therefrom.
The above pharmaceutical compositions may also be used in immunotherapy methods for treating a subject having cancer. For use in immunotherapy, the present invention further provides a third pharmaceutical composition comprising antibodies directed against Int6 protein or peptides derived therefrom where such antibodies are coupled to toxin molecules, radioisotopes or drugs.
The present invention therefore relates to application of immunotherapy to subjects having cancer comprising administering one or more of the above pharmaceutical compositions to said subject in a therapeutically effective amount.
The invention further relates to a method for treating a subject having cancer comprising:
(a) immunizing the subject with an amount of an expression vector encoding Int6 protein or with Int6 protein itself, said amount effective to elicit a specific T cell response;
(b) isolating said T cells from said immunized subject; and
(c) administering said T cells to said immunized subject or to an unimmunized subject in a therapeutically effective amount.
The invention also provides a diagnostic kit for determining the nucleotide sequence of Int6 alleles by the polymerase chain reaction, said kit comprising purified and isolated nucleic acid sequences useful as PCR primers. These PCR primers are also useful in analyzing DNA or RNA of a subject for mutations of the Int6 gene.
The present invention further provides a method for supplying the wild-type Int6 gene to a cell having altered expression of the Int6 protein by virtue of a mutation in the Int6 gene, the method comprising: introducing a wild-type Int6 gene into a cell having altered expression of the Int6 protein such that said wild-type gene is expressed in the cell.